The detection and enumeration of microorganisms is practiced in numerous settings, including the food-processing industry (testing for the contamination of food by microorganisms such as E. coli and S. aureus), the health care industry (testing of patient samples and other clinical samples for infection or contamination), environmental testing industry, the pharmaceutical industry, and the cosmetic industry.
Growth-based detection and enumeration of microorganisms is commonly practiced using either liquid nutrient media (for example, most probable number analysis (MPN)) or semi-solid nutrient media (agar petri dishes). Enumeration using the liquid MPN method is typically achieved by placing serial 10-fold dilutions of a sample of interest in replicate sets of tubes containing selective media and chemical indicators. The tubes are incubated for 24-48 hours at elevated temperatures (30-37.degree. C.) followed by examination for growth of organisms. A statistical formula, based on the volume of sample tested and the number of positive and negative tubes for each set, is used to estimate the number of organisms present in the initial sample.
This method of performing MPN analysis has several disadvantages. It is labor intensive because of the multiple diluting and pipetting steps necessary to perform the analysis. Typically, it is only practical to use replicate sets of about three to five tubes for each dilution. As a result, the 95% confidence limits for an MPN estimate for microbial concentration are extremely wide. For example, a three tube MPN estimate of 20 has 95% confidence limits ranging from 7 to 89. Furthermore, results typically are not obtainable in less than twenty-four hours.
Multi-well devices have been introduced for use in connection with MPN analysis. A user inoculates the device by introducing a sample of the item to be tested, such as food, onto the substrate containing the wells. Typically, the sample includes growth media and indicators. Once inoculated, the user incubates the device and then calculates the MPN based on the number of "positive" wells.
Multi-well devices present many potential problems. Inoculation may be hampered by air bubbles that form in the wells during introduction of the sample. Each well may not receive the same volume of sample. Furthermore, the method of inoculation or the device may promote bridging and cross-contamination among the wells, thereby potentially adversely affecting the MPN calculation.
Another potential problem with multi-well devices is that they may be inconvenient to use. For example, most multi-well devices are inoculated by either pipetting sample directly into the wells or pouring sample onto the multi-well substrate. Pippetting is labor and time intensive. Pouring requires that the wells be evenly filled and that excess sample be poured off. In either event, these devices may lend themselves to contamination by outside sources during the inoculation procedure.